Vaccinia virus presents a model system in which to study viral multiplication, regulatory mechanisms, and pathogenesis. As part of the continuing effort in this laboratory to use a genetic approach to study viral replication, we have isolated 40 temperature-sensitive (ts) mutants of vaccinia virus. This proposal addresses the phenotypic and genetic characterization of these mutants. The phenotypic characterization of a series of ts mutants has two goals. The first is to identify specific classes of mutants that can be used as tools to study various aspects of vaccinia virus replication that have proved refractory to biochemical analyses. The second goal is to allow the construction of a functional map in conjunction with recombination experiments. The specific characteristics that will be examined include: (1) the pattern of polypeptide synthesis in cells infected at the nonpermissive temperature as revealed by SDS-polyacrylamide gel electrophoresis, (2) the formation of intermediates in virion assembly as visualized by electron microscopy, (3) the ability to synthesize viral DNA as measured by (3H) thymidine incorporation, (4) the thermolability of virion infectivity, and (5) the thermolability and temperature-sensitivity of the virion-associated enzyme activities. In many viral systems, knowledge of the functional organization of the genome has revealed the mechanisms by which viral replication is regulated. Recombination experiments with standard 2- and 3-factor crosses will be performed to construct a functional map of the 40 ts mutants already isolated. In addition, the feasibility of marker rescue experiments, which would allow the alignment of the genetic map with the physical map obtained by restriction endonuclease digestion of vaccinia DNA, will be examined.